CN105723221B - Separation method, detection method, signal measuring method, the determination method of disease, the method for evaluating drug effect of disease curative, kit and fluid composition - Google Patents
Separation method, detection method, signal measuring method, the determination method of disease, the method for evaluating drug effect of disease curative, kit and fluid composition Download PDFInfo
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Abstract
The present invention provide field trash removal efficiency is excellent and vesica with bilayer lipid membrane is not easy the separation method of the destroyed vesica with bilayer lipid membrane.The separation method of the above-mentioned vesica with bilayer lipid membrane is characterized in that, comprising:Complex formation process makes the organism sample containing the vesica with bilayer lipid membrane be contacted with being bonded with the solid phase carrier for the ligand for identifying the surface antigen for being present in above-mentioned vesicle surface, forms the complex of above-mentioned vesica and above-mentioned solid phase carrier;Cleaning process cleans above-mentioned complex;At least any process in above-mentioned complex formation process and above-mentioned cleaning process is carried out in the presence of nonionic surfactant.
Description
Technical field
The present invention relates to separation method, detection method, signal measuring method, the determination method of disease, disease curatives
Method of evaluating drug effect, kit and fluid composition.More specifically, it is related to the vesica that excretion body etc. has bilayer lipid membrane
Separation method, the judgement side of the nucleic acid of this method or the detection method of protein, signal measuring method, disease is utilized
Make in method, the method for evaluating drug effect of disease curative, disease judgement or evaluating drug effect kit and above-mentioned separation method
Fluid composition.
Background technology
Vesica has the structure surrounded of bilayer lipid membrane, in such vesica, as existing in the body fluid in organism
Vesica particle, it is known to excretion body.It is known on the surface of excretion body, with general cell surface similarly, the presence of various film eggs
On the other hand white matter, will also realize that in the inside of excretion body, also contain microRNA in addition to the various protein such as cell factor
(miRNA)。
Additionally, it is known that the various cells secretion excretion bodies such as cell, various cancer cells from immune system, as in organism
Cell-cell communication instrumentality excretion body function, the related receptors of the diseases such as excretion body and physiological phenomenon, cancer is to closing
Note, is carrying out these researchs.For example, suffer from it is reported that during antibody used as the EpCAM of tumor markers from oophoroma
Excretion body is detached in the blood circulation of person, there is correlation between progress of the miRNA expression quantity from excretion body with oophoroma
(non-patent literature 1).
In addition, as the memebrane protein of tetratransmembrane expressed on excretion body, have and belong to tetratransmembrane albumen family
CD9, CD63 and CD81, the excretion scale of construction reported in non-patent literature 2 in the blood plasma of melanoma patients is higher than Healthy People,
This can with for CD63 antibody, for the antibody of the Caveolin-1 of tumor related marker object be detected quantification.
In addition, CD63 antibody is combined to the plasma sample after centrifugation, is reacted for antibody of various memebrane proteins etc., thus
Quantitative resolution (patent document 1) is carried out to the signal of the excretion body from cancer patient.
Existing technical literature
Patent document
Patent document 1:International Publication No. 2010/065968
Non-patent literature
Non-patent literature 1:D.D.Taylor.,et al.,Gynecol.Oncol.,110,13-21(2008)
Non-patent literature 2:M.Logozzi.,et al.,PLoS ONE.,4,1-10(2009)
Invention content
However, reaction solution, cleaning solution, the buffer solution that antibody linked particle suspends as the solid phase carrier for being bonded antibody
Function, it is desirable that reduce the non-specific adsorption to the reaction vessels such as tablet, particle surface, reduce the particle that occurs in reaction
The function of cohesion.
However, the particle as magnetic particle captures the vesica covered as above-mentioned excretion body by bilayer lipid membrane
When, above-mentioned non-specific adsorption is not only easily caused, and easily generate the mutual cohesion of particle, sometimes to the recycling of vesica
Rate, the cleaning efficiency of particle adversely affect.If the cleaning of the antibody linked particle after reaction is insufficient, exists and come from
The problem of protein that is mingled with of a specimen is mixed into, and the refined degree of excretion body reduces.
Therefore, when the vesica covered by bilayer lipid membrane is detached, it is also desirable to the non-spy to solid phase carrier can be reduced
The opposite sex absorption buffer solution high with the cohesion of particle, cleaning ability.
However it has been found that it even if will be generally used for that the protein such as non-specific adsorption, BSA used in particle coacervation is inhibited to add
It is added to particle to preserve in buffer solution, reaction buffer etc., when the vesica covered by bilayer lipid membrane is detached, particle can also coagulate
It is poly-, and then Proteome Analysis of refined excretion body followed by etc. is impacted.
In addition, when the vesica covered by bilayer lipid membrane is detached, also require to the broken of the vesica with bilayer lipid membrane
It is bad few.
The problem to be solved by the present invention is that the removal efficiency for providing field trash is excellent and has the vesica of bilayer lipid membrane
It is not easy the separation method of the destroyed vesica with bilayer lipid membrane.
Therefore, the inventors of the present invention further investigate, as a result, it has been found that by including complex formation process and cleaning process
In the separation method of vesica with bilayer lipid membrane, the above-mentioned compound bodily form is carried out in the presence of nonionic surfactant
Into at least any process in process and above-mentioned cleaning process, the above subject can be solved, so as to complete the present invention, wherein,
Complex formation process be make containing with bilayer lipid membrane vesica organism sample be bonded with identification be present in it is above-mentioned
The solid phase carrier contact of the ligand of the surface antigen of vesicle surface forms the complex of above-mentioned vesica and above-mentioned solid phase carrier;Clearly
It is the above-mentioned complex of cleaning to wash process.
That is, the present invention provides a kind of separation methods of the vesica with bilayer lipid membrane of 1 > of <, which is characterized in that including:
Resist the surface that the organism sample containing the vesica with bilayer lipid membrane is present in above-mentioned vesicle surface with being bonded with identification
The solid phase carrier of former ligand is contacted, and the complex for forming the complex of above-mentioned vesica and above-mentioned solid phase carrier forms work
Sequence;With the cleaning process for cleaning above-mentioned complex;Above-mentioned complex is carried out in the presence of nonionic surfactant to be formed
At least any process in process and above-mentioned cleaning process.
In addition, the present invention provides the detection method of the nucleic acid in a kind of vesicas of 2 > of <, which is characterized in that in above-mentioned 1 > of <
Separation method after, further include detection vesica in nucleic acid detection of nucleic acids process.
And then the present invention provides a kind of detection methods of the protein from vesica of 3 > of <, which is characterized in that above-mentioned
After the separation method of 1 > of <, further include and protein existing for at least one party in the inside of vesica and surface is carried out
The protein detection process of detection.
And then the present invention provides a kind of signal measuring methods from vesica of 4 > of <, which is characterized in that in above-mentioned 1 > of <
Separation method after, further include the signal that the signal strength of the vesica to carrying out the above-mentioned complex of self-forming is measured and survey
Determine process.
And then the present invention provides a kind of determination methods of disease of 5 > of <, which is characterized in that is whether judgement examinee sends out
The method of disease, comprising the organism sample from examinee is used, using the signal measuring method of above-mentioned 4 > of < to carrying out self-forming
The process that the signal strength of the vesica of above-mentioned complex is measured.
And then the present invention provides a kind of method of evaluating drug effect of disease curative of 6 > of <, which is characterized in that is that disease is controlled
The method of evaluating drug effect of medicine is treated, comprising being tried using the organism from examinee before the giving of disease curative and after giving
Sample is measured the signal strength for carrying out the vesica of the above-mentioned complex of self-forming using the signal measuring method of above-mentioned 4 > of <
Process.
And then the present invention provides a kind of kits of 7 > of <, which is characterized in that has and is bonded with identification and is present in fat
The solid phase carrier of the ligand of the surface antigen on the surface of the vesica of matter duplicature and the liquid containing nonionic surfactant
Composition.
And then the present invention provides a kind of fluid compositions of 8 > of <, which is characterized in that containing nonionic surfactant,
It is used in the separation method of the vesica with bilayer lipid membrane, it is above-mentioned that there is bilayer lipid membrane for cleaning above-mentioned complex
The separation method of vesica include:The organism sample containing the vesica with bilayer lipid membrane is made to be present in being bonded with identification
The solid phase carrier contact of the ligand of the surface antigen of above-mentioned vesicle surface forms the complex of above-mentioned vesica and above-mentioned solid phase carrier
Complex formation process;Clean the cleaning process of above-mentioned complex.
For the separation method of the vesica with bilayer lipid membrane of the present invention, the removal efficiency of field trash is excellent, and
And the vesica with bilayer lipid membrane is not easy to be destroyed.
In addition, when the kit of the present invention, fluid composition are used for the separation of the vesica with bilayer lipid membrane, it is mingled with
Object is fully removed, also, the vesica with bilayer lipid membrane is not easy to be destroyed.
Description of the drawings
Fig. 1 is the antibody and the influence reacted of excretion body for representing nonionic surfactant para-linkage in particle
Figure.
Fig. 2 is the silver staining color image for the cleaning ability for representing the cleaning buffer solution containing nonionic surfactant.
Fig. 3 is to represent that the cleaning buffer solution containing nonionic surfactant is difficult to remove from the antibody for being bonded to particle
The western blot image of excretion body.
Fig. 4 is the unquestioned grain of grain size of excretion body for representing to be captured with the magnetic particle for being bonded 3 antibody of anti-CD 6
Spend the waveform of distribution.
Fig. 5 is the unquestioned size distribution of grain size for the excretion body for representing to be captured with each antibody linked magnetic particle
Figure.
Fig. 6 is the form transmission-type that there is no problem the electricity for the excretion body for representing to be captured with anti-CD9 antibody linkeds magnetic particle
Sub- microscopic iage.
Fig. 7 is the Biochemical Analyzer for representing to detect nucleic acid with antibody linked magnetic particle from the excretion body captured
Analysis diagram.
Fig. 8 is that represent can be to carrying out quantitative expansion from the microRNA in the excretion body that antibody linked magnetic particle captures
Increase curve.
Fig. 9 is to represent that inside and surface from vesica can be detected from the vesica captured with antibody linked magnetic particle
The western blot image of protein.
Figure 10 be the vesica for representing to obtain using antibody linked magnetic particle capture amount and the amount of vesica reacted into than
The western blot image of example.
Figure 11 is to represent to detect to capture from various culture supernatants and body fluid come antibody linked magnetic particle of using by oneself
Vesica surface protein western blot image.
Specific embodiment
(separation method with the vesica of bilayer lipid membrane)
The separation method of the vesica with bilayer lipid membrane of the present invention, which is characterized in that include:Make containing with lipid
The organism sample of the vesica of duplicature and the solid phase for being bonded with the ligand for identifying the surface antigen for being present in above-mentioned vesicle surface
Carrier contacts, and forms the complex formation process of the complex of above-mentioned vesica and above-mentioned solid phase carrier;With the above-mentioned complex of cleaning
Cleaning process, carried out in the presence of nonionic surfactant in above-mentioned complex formation process and above-mentioned cleaning process
At least any process.
(nonionic surfactant)
The nonionic surfactant used in separation method as the present invention, does not preferably contain fragrance in the molecule
Race's group.By using such nonionic surfactant for not containing aromatic group in the molecule, so as to
It does not destroy in the case of forming the bilayer lipid membranes of vesicas such as excretion body, reduces field trash and the non-specificity of solid phase carrier is inhaled
It is attached.In addition, the reactivity of the catching reaction of vesica and solid phase carrier improves.Also, it can also inhibit refined excretion body to egg
The influence of white matter group parsing etc..
As the above-mentioned nonionic surfactant for not containing aromatic group in the molecule, polyethylene glycol can be enumerated
Type nonionic surfactant, to the polyalcohols such as glycerine, pentaerythrite add in hydrophobic group obtained by polyol type it is non-from
Sub- property surfactant.
As above-mentioned polyethylene glycol type nonionic surfactant, as long as with polyethylene oxide chain as hydrophily
Group, there is no particular limitation, polyalkylene glycol ethylene oxide adduct can be enumerated, polyol fatty acid ester ethylene oxide adds
Into object, higher alcohol ethylene oxide adduct, fatty acid ethylene oxide addition product, senior alkylamines ethylene oxide adduct, fat
The ethylene oxide adduct etc. of sour amide epoxy ethane additive product, grease.
Wherein, from non-specific adsorption minimizing effect, it is few to the damage of vesica from the aspect of, preferred polyalkylene glycol ring
Oxidative ethane addition product, polyol fatty acid ester ethylene oxide adduct.
As above-mentioned polyalkylene glycol ethylene oxide adduct, preferably block copolymerization type, more preferably have by polycyclic oxygen
The block (hereinafter also referred to as block A) and be 3 by the carbon atom number of above-mentioned alkylidene that polyoxyalkylene is formed that ethane is formed
The block copolymer of above block (hereinafter also referred to as B block).In addition, as the carbon of alkylidene oxygroup contained in B block
Atomicity, preferably 3~6, more preferable 3 or 4, particularly preferred 3.It should be explained that the carbon of the alkylidene oxygroup contained in B block is former
The polyalkylene glycol ethylene oxide adduct that subnumber is 3 is polypropylene glycol ethylene oxide adduct.
In addition, total content as block A, in block copolymer, preferably 15~99 mass %, more preferable 50~99
Quality %, further preferred 60~95 mass %, particularly preferred 70~90 mass %.
In addition, total content as B block, from the viewpoint of cytotoxicity, in block copolymer, preferably 1~
85 mass %, more preferable 1~50 mass %, further preferred 5~40 mass %, particularly preferred 10~30 mass %.
It should be explained that these contents can be measured with NMR etc., when block A is 2 or more, 2 or more embedding is represented
The aggregate value of each content of section A.It is the aggregate value of each content of the B block of 2 or more when similarly B block is 2 or more.
In such polyalkylene glycol ethylene oxide adduct, preferred pluronic type nonionic surfactant etc.
The ABA type triblock copolymer with the structure that B block is clipped with 2 block A as illustrated.
As the commercially available product of polyalkylene glycol ethylene oxide adduct, for example, can enumerate Pluronic F-68,
Pluronic L-62 (being ADEKA systems) etc..
In addition, as above-mentioned polyol fatty acid ester ethylene oxide adduct, the preferred ring of sorbitan fatty acid ester
The ethylene oxide adduct of oxidative ethane addition product, sorbitan fatty acid ester, the epoxy of more preferable sorbitan fatty acid ester
Ethane additive product.
In addition, " aliphatic acid " in polyol fatty acid ester ethylene oxide adduct can be unrighted acid, saturation
Any one of aliphatic acid.In addition, aliphatic acid can be straight-chain or branched, but preferably straight-chain.In addition,
The carbon atom number of aliphatic acid preferably 8~18, more preferably 10~20, further preferably 12~18.Specifically, the moon can be enumerated
Cinnamic acid, palmitic acid, stearic acid, oleic acid etc..
As the commercially available product of polyol fatty acid ester ethylene oxide adduct, for example, can enumerate Tween20, Tween40,
Tween60, Tween65, Tween80, Tween85 (being and the pure medicine system of light) etc..
In addition, as polyol type nonionic surfactant obtained by adding in hydrophobic group to polyalcohol, can lift
Go out the aliphatic ester of the aliphatic ester of glycerine, the aliphatic ester of pentaerythrite, D-sorbite or sorbitan, the fat of sucrose
Fat acid esters, the alkyl ether of polyalcohol, ethyl alcohol amine fatty acid amide etc..Wherein, preferably with fatty acid residue as hydrophobic
The polyol type nonionic surfactant of property group.It should be explained that fatty acid residue preferably with above-mentioned polyol fatty acid
The identical group of the fatty acid residue that contains in ester ethylene oxide adduct.
In polyol type nonionic surfactant with such fatty acid residue, preferably D-sorbite or mountain
The aliphatic ester of pears sugar alcohol acid anhydride, more preferable sorbitan fatty acid ester.
In addition, in the above-mentioned nonionic surfactant for not containing aromatic group in the molecule, from non-specificity
Adsorb minimizing effect, it is few to the damage of vesica from the aspect of, preferred polyethylene glycol type nonionic surfactant is more preferably poly-
Aklylene glycol ethylene oxide adduct, polyol fatty acid ester ethylene oxide adduct, further preferred polyalkylene glycol
Ethylene oxide adduct.
It should be explained that nonionic surfactant can be used alone, two or more use can also be combined.
In addition, the HLB of the nonionic surfactant other than polyalkylene glycol ethylene oxide adduct
(Hydrophile-Lipophile Balance) value is preferably more than 13.1, and more preferable more than 13.5.
It should be explained that in this specification, HLB value represents the hydrophilic lipophilic balance of Griffin, represents surfactant pair
Water, oil compatibility degree value.For example, the HLB value that the HLB value of Tween20 is 16.7, Tween80 is 15.0.In addition,
The HLB value for belonging to the TritonX-100 of the nonionic surfactant containing aromatic group is 13.5, Nonidet P-
40 HLB value is 13.1.
In addition, from the viewpoint of cytotoxicity, as the weight average molecular weight of nonionic surfactant, preferably 500
~50000, more preferable 1000~30000, further preferred 2000~20000.
It should be explained that weight average molecular weight can use the measure such as liquid chromatogram, NMR, MALDI-TOF/MS.
The concentration of nonionic surfactant is relative to the total amount of the liquid phase in system (from organism sample, buffer solution
Deng total measurement (volume) in subtract the amounts of the solid constituents such as solid phase carrier) with final concentration, more than preferably 0.005% (w/v) is more excellent
It is selected as more than 0.01% (w/v), more than further preferably 0.015% (w/v), more than particularly preferably 0.02% (w/v), separately
Outside, preferably 10% (w/v) hereinafter, more preferably 5% (w/v) hereinafter, further preferably 3.5% (w/v) hereinafter, especially excellent
It is selected as 2% (w/v) below.It should be explained that final concentration preferably above-mentioned in complex formation process, cleaning process.
If the concentration of nonionic surfactant, in the range of above-mentioned numerical value, non-specific adsorption reduces effect
Fruit is excellent, and solid phase carrier is excellent to prevent the effect of cohesion in the case of particle.In addition, it does not hinder between ligand and surface antigen
Combination.
In addition, in the present invention, complex formation process and clear can be carried out in the presence of nonionic surfactant
Any process in process is washed, complex formation process and cleaning can also be carried out in the presence of nonionic surfactant
This two side of process.Furthermore it is possible to before complex formation process, in advance by the buffer solution containing nonionic surfactant
Compositions is waited to be added in organism sample and use.
Specifically, in the case that solid phase carrier is tablet, threadiness is not dispersed in the shape in liquid phase, nonionic
Surfactant can add in liquid phase when complex formation process, in the form of the cleaning solution that is used in cleaning process is medium
Into system.In addition, in the case that solid phase carrier is with particle shape dispersion in the liquid phase as magnetic particle, preferably addition in advance
It is used into the particle dispersion before the organism sample addition as a specimen or is added to the slow of adjustment organism sample
The cleaning solution used in fliud flushing, in cleaning process is medium.
For example, by adding nonionic surfactant in the preservation buffer solution of antibody linked particle, with BSA, its
His polymer phase ratio improves the dispersibility of the solid phase carrier of particle shape, can reduce cohesion and to preserving container, reaction vessel
Non-specific adsorption.It, can be not destroy animal thin in addition, by adding nonionic surfactant in cleaning buffer solution
The absorption of the non-specific ingredient from a specimen is substantially reduced in the case of the vesicas such as born of the same parents, excretion body, using not containing in the molecule
During the nonionic surfactant of aromatic group, the cleaning ability of antibody linked particle can be also increased to and as tool
The level for having the Triton X-100 of the nonionic surfactant of aromatic group equal.
(complex formation process)
Complex formation process is that the organism sample containing the vesica with bilayer lipid membrane is made to be deposited with being bonded with identification
It is the solid phase carrier contact of the ligand of the surface antigen of vesicle surface, the process for forming the complex of vesica and solid phase carrier.
By above-mentioned contact, vesica is captured by ligand, forms the complex of vesica and solid phase carrier.It should be explained that complex forms work
In the reaction system of sequence, other than the ligand being bonded with above-mentioned solid phase carrier, identification can also coexist and be present in lipid pair
The ligand of the surface antigen on the surface of the vesica of tunic.
As long as above-mentioned organism sample contains the vesica with bilayer lipid membrane, there is no particular limitation, for example, can lift
Go out the various liquid such as body fluid, thalline liquid, the culture medium of cell culture, cell culture supernatant, histiocytic crushing liquid.Its
In, preferably body fluid, cell culture supernatant.As body fluid, can enumerate whole blood, serum, blood plasma, blood constituent, various haemocytes,
The blood such as clot, blood platelet constituent and urine, sperm, breast milk, sweat, interstitial fluid, chromic fibrous lymph, bone marrow fluid, tissue
Liquid, saliva, gastric juice, joint fluid, liquor pleurae, bile, ascites, amniotic fluid etc., preferably blood constituent, urine.It is using the present invention
Separation method selectively and efficiently can isolate vesica from broad category of organism sample.For example, use blood plasma, blood
When being used as organism sample clearly, it is not easy to that non-specific adsorption occurs.
It should be explained that blood constituent can be handled with anti-coagulants such as citric acid, heparin, EDTA.
Above-mentioned organism sample can in advance be added in buffer composition containing nonionic surfactant etc.
It carries out pre- previously processed and uses, but can also directly use the sample taken from organism.That is, separation side using the present invention
Method, also can be without easily being selected using the PEG precipitation method, using the pre-treatment of the partition method of ultracentrifuge etc. etc.
Property separation.
In addition, as the above-mentioned vesica with bilayer lipid membrane, cell can be enumerated, from cell to the excretion extracellularly released
Vesica as body, separation method of the invention is particularly preferred for the situation that vesica is excretion body.In general, solid phase carrier
In the case of for particle shape as magnetic particle, during via the antibody capture excretion body for being bonded to particle, particle is easy each other
Cohesion, the redisperse of particle becomes difficult, therefore the flushing of field trash sometimes becomes difficult, but in the present invention, can be by adding
Add nonionic surfactant to reduce the mutual cohesion of particle.
As long as in addition, in surface antigen existing for the surface of vesica in substance existing for the surface of vesica and with antigen
Property, just it is not particularly limited.For the surface antigen for enumerating excretion body, the four transmembrane proteins such as CD9, CD63, CD81 can be enumerated and surpassed
The same clan of family;The antigen presentations GAP-associated protein GAP such as MHCI, MHCII;Integrin, ICAM-1, EpCAM etc. are bonded molecule;EGFRvIII、
The cell factors such as TGF-β/cytokine receptor, enzyme etc..Wherein, it is preferably in the antigen protein in excretion body surface face.
In addition, the usage amount of above-mentioned organism sample (from organism sample, is buffered relative to the total amount of the liquid phase in system
The amount of the solid constituents such as solid phase carrier is subtracted in the total measurement (volume) of liquid etc.) with final concentration, preferably 1~90% (w/v), more preferable 10
~50% (w/v).
In addition, as long as the solid phase carrier used in the separation method of the present invention, which is bonded with identification, is present in vesicle surface
Surface antigen ligand solid phase carrier, be just not particularly limited.
As ligand, preferably identification is present in the antibody of the surface antigen of vesicle surface, and more preferably identification is present in excretion
The antibody of the antigen protein in body surface face.Furthermore it is possible to it is monoclonal antibody or polyclonal antibody, but preferred Dan Ke
Grand antibody.
Said monoclonal antibody is not particularly limited, can according to well known method, such as K.Watanabe et al.,
Vasohibin as an endothelium-derived negative feedback regulator of
It is prepared by angiogenesis, J.Clin.Invest.114 (2004), the method described in 898-907.In addition, identification excretion body
The monoclonal antibody of the four transmembrane proteins superfamily class such as surface antigen CD9, CD63, CD81 be referred to International Publication No.
The manufactures such as No. 2013/099925 bulletin.
It should be explained that the type as above-mentioned antibody, can enumerate IgG, IgM, preferably IgG.Further, it is possible to use by them
Degraded segment.For example, F (ab ') 2, Fab ', Fab etc. can be enumerated.
As the material for the solid phase carrier for being bonded above-mentioned ligand, for example, polystyrene type, polyethylene kind, poly- third can be enumerated
Alkenes, polyesters, poly- (methyl) vinyl cyanide, styrene-butadiene copolymer, poly- (methyl) esters of acrylic acid, fluororesin, friendship
Join the high-molecular compounds such as glucan, polysaccharide;Glass;Metal;Magnetic substance;Resin combination containing magnetic substance;Their group
Close etc..
In addition, the shape of solid phase carrier is not particularly limited, for example, can enumerate hypocrateriform, spherical, particle shape, threadiness,
Rodlike, plate-like, container-like, pond (セ Le), minitype plate, developmental tube etc..
In the present invention, from the viewpoint of separation of solid and liquid, the easiness of cleaning, preferred magnetic particles.
As above-mentioned magnetic particle, for example, ferroso-ferric oxide (Fe can be enumerated3O4), di-iron trioxide (γ-Fe2O3), it is each
The metals such as kind ferrite, iron, manganese, nickel, cobalt, chromium;The fine magnetic-substance particle being made of the alloy of cobalt, nickel, manganese etc.;Contain this in resin
The magnetic particle of a little magnetic substances.As resin, hydrophobic polymer, hydrophilic polymer etc. can be enumerated.
Wherein, the magnetic particle containing magnetic substance in preferred resin, more preferably in the master batch containing super paramagnetic microsphere
Surface is formed with the magnetic particle of polymeric layer.For example, can enumerate Japanese Unexamined Patent Publication 2008-32411 bulletins record by
The surface of master batch containing super paramagnetic microsphere forms hydrophobic 1st polymeric layer, is formed at least on the 1st polymeric layer
There is the 2nd polymeric layer of glycidyl on surface, which is chemically modified and has imported polar group
Magnetic particle.
Here, as super paramagnetic microsphere, representative is the iron oxide of (5~20nm of preferable particle size) below grain size 20nm
The particle of system can be enumerated by XFe2O4(X=Mn, Co, Ni, Mg, Cu, Li0.5Fe0.5Deng) represent ferrite, by Fe3O4It represents
Magnetic iron ore, γ-Fe2O3, from the aspect of and remanent magnetization strong from saturated magnetization is few, preferably comprise γ-Fe2O3And Fe3O4
In either one.
In addition, being used to form the monomer of above-mentioned hydrophobic 1st polymeric layer, it is roughly divided into mono-functional's monomer, crosslinking
Property monomer.
As above-mentioned mono-functional's monomer, for example, may be exemplified styrene, α-methylstyrene, halogenated styrenes etc.
Aromatic vinyl base system monomer;Methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, acrylic acid
Stearyl ester, stearyl methacrylate, cyclohexyl acrylate, cyclohexyl methacrylate, isobornyl acrylate, metering system
The ethylenic unsaturated carboxylic acids Arrcostab such as sour isobornyl thiocyanoacetate system monomer.
In addition, as above-mentioned cross-linkable monomer, glycol diacrylate, ethyleneglycol dimethacrylate may be exemplified
Ester, trimethylolpropane trimethacrylate, trimethylol-propane trimethacrylate, pentaerythritol triacrylate, season penta
Multi-functional (the first such as tetrol trimethyl acrylic ester, dipentaerythritol hexaacrylate, dipentaerythritol hexamethacrylate
Base) acrylate;Butadiene, isoprene equiconjugate diene and divinylbenzene, diallyl phthalate, acrylic acid
Allyl ester, allyl methacrylate etc..
In addition, the monomer of above-mentioned 2nd polymeric layer is used to form to import functional group as the main purpose to particle surface, packet
Include the monomer containing glycidyl.As the content of the monomer containing glycidyl, it is being used to form the 2nd polymeric layer
In monomer, preferably more than 20 mass %.Here, as the co-polymerized monomer containing glycidyl, acrylic acid shrink can be enumerated
Glyceride, glycidyl methacrylate, allyl glycidyl ether etc..
The polar group for being chemically modified and importing as the glycidyl to the 2nd polymeric layer, preferably can with
The functional group of precursor reactant, further preferably 1 or more at least one kind of atom in oxygen atom, nitrogen-atoms and sulphur atom.Its
In, more preferable amino, aldehyde radical, carboxyl, active ester groups.Particularly the 2nd polymeric layer of magnetic particle has above-mentioned polar group
It is binding affinity good with ligand during with 2,3- dihydroxypropyls.
As the method for being bonded ligand with solid phase carrier, physisorphtion, covalent bond method, ionic bond method etc can be used
The method of chemical bonding etc..As physisorphtion, it can enumerate and ligand is directly fixed on the method for solid phase carrier and white egg
It is adsorbed and fixed method etc. after waiting the bonding of other protein chemistries in vain.As the method for chemical bonding, it can enumerate and utilize importing
To surface of solid phase carriers can be with the method for functional group's Direct Bonding of ligand reaction on solid phase carrier;In solid phase carrier with matching
The method for importing the laggard line unit conjunction of spacer molecule (carbodiimide compound etc.) between body with chemical bond;Make ligand and albumin etc.
After other protein bonds, by method of the protein and solid phase carrier chemical bonding etc..
In addition, the usage amount of the solid phase carrier of above-mentioned ligand is bonded with relative to the total amount of the liquid phase in system (from biology
The total measurement (volume) of body sample, buffer solution etc. subtracts the amount of the solid constituents such as solid phase carrier) with final concentration, preferably 0.005~5%
(w/v), more preferable 0.01~1% (w/v).
Complex formation process other than above-mentioned each ingredient, can also use the albumen such as salt, albumin as needed
Surfactant other than matter, aforementioned nonionic surfactant etc., but if the analysis etc. after considering, then preferably not
Use protein, nucleic acid.
Reaction temperature in complex formation process is usually in the range of 2~42 DEG C, and the reaction time is usually 5 minutes~
24 hours.When being reacted for 2~8 DEG C, the reaction time is preferably 8~30 hours or so, in room temperature (20 DEG C)~42 DEG C of progress
During reaction, the reaction time is preferably 5~60 minutes or so.
In complex formation process, the pH in system is not particularly limited, but the preferably range of pH5~10, more preferably
Range for pH6~8.In order to maintain target pH, usually using buffer solution, for example, phosphate buffer, three (methylols) can be enumerated
Aminomethane buffer solution, HEPES buffer solution, MES buffer solutions etc..
(cleaning process)
Cleaning process is that the vesica and the complex of solid phase carrier that are formed in above-mentioned complex formation process are cleaned
Process.Unreacted ingredient, unreacted mark substance etc. are removed as a result,.It can directly clean in complex formation process
The system containing complex.
Above-mentioned cleaning process is divided into two kinds generally according to the shape of solid phase carrier.The solid phase carrier as magnetic particle is grain
During sub- shape, can enumerate for example makes magnetic particle be dispersed in the method cleaned in cleaning solution, and on the other hand, solid phase carrier is micro-
As orifice plate during form, can enumerate makes cleaning solution contact the method that its surface is cleaned.Whether any state, this hair
In bright, as cleaning solution, it is preferable to use containing the above-mentioned nonionic surfactant for not containing aromatic group in the molecule
Cleaning solution.
In addition, when solid phase carrier is magnetic particle, as cleaning process, preferably comprises and assembled magnetic particle by magnetic force
And make the collection magnetic process of magnetic particle and liquid phase separation and the magnetic particle detached in the collection magnetic process is made to be dispersed in cleaning solution
Dispersion step.Thereby, it is possible to from magnetic particle surface more efficiently by the field trash in unreacted substance, organism sample
It cleans, be separated off.
Specifically, following carry out:Magnetic fields is made magnetic particle to be made to be attached to reactor vessel wall in reaction vessel
And after assembling, except dereaction supernatant, cleaning solution is further added in as needed, and supernatant is removed after similarly making magnetic fields
Liquid, repeatedly aforesaid operations.
As cleaning solution, preferably comprise the above-mentioned nonionic surfactant for not containing aromatic group in the molecule and
The cleaning solution of buffer solution enumerated in above-mentioned complex formation process.The pH of cleaning solution is preferably the range of pH5~10, more preferably
Range for pH6~8.Specifically, TBS containing 0.01% nonionic surfactant etc. can be enumerated.
(dissociation process)
The separation method of the present invention, can also be with the vesica being caught in from dissociation of ligand after above-mentioned cleaning process
Dissociate process.
Ligand be specific antibody when, as by caused by antigen-antibody reaction specific binding dissociate condition,
Know with the various dissociation of the progress such as affinity chromatography (for example, referring to " Afinity Chromatography principles&
methods”Pharmacia LKB Biotechnology).Above-mentioned dissociation process can carry out accordingly.That is, as in the present invention
The dissociation solution used can enumerate the acid solutions such as hydrochloric acid, sulfuric acid, propionic acid, acetic acid, glycine/hydrochloride buffer;Sodium hydroxide water
The alkaline solutions such as solution, potassium hydroxide aqueous solution, ammonia spirit, diethylamine;3M sodium-chloride water solutions, 4.5M magnesium chloride brines
Wait high inonic strength solutions;Solution containing surfactants such as SDS, TritonX-100, Tween20;Contain twoAlkane, second
Glycol etc. reduces the buffer solution of the substance of polarity;Contain the slow of the chaotropics such as trichloroacetic acid, thiocyanic acid ion and urea, guanidine hydrochloride etc.
Fliud flushing.
(detection method of nucleic acid and the detection method of protein)
The detection method of nucleic acid in the vesica of the present invention is after above-mentioned separation method, is further included in vesica
The detection of nucleic acids process that is detected of nucleic acid.
In addition, the detection method of the protein from vesica of the present invention is after above-mentioned separation method, further wrap
Containing the protein detection process being detected to protein existing for at least one party in the inside of vesica and surface.
These detection methods carry out according to conventional methods other than carrying out above-mentioned separation method.It is above-mentioned that there is fat
When the vesica of matter duplicature is excretion body, according to known in PCR methods, electrophoresis, western blot method, immunochemical method etc.
Method survey nucleic acid, protein from the excretion physical examination of recycling.In addition, as nucleic acid, miRNA, mRNA can be enumerated.
Particularly, cell, various cancer cells secrete of the excretion body from various cells such as immune system, so using this hair
The detection method of bright nucleic acid detects the nucleic acid (particularly miRNA etc.) from excretion body, it is parsed, thus, it is possible to
Carry out the judgement of physiological phenomenon, various diseases.
(signal measuring method)
The signal measuring method from vesica of the present invention is after above-mentioned separation method, is further included to carrying out idiomorphism
The signal measuring process being measured into the signal strength of the vesica of above-mentioned complex.
The measure of above-mentioned signal strength is carried out according to immunoassay, for example, enzyme immunoassay can be enumerated
(EIA), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), radioactive ray immunoassay (RIA), shine exempt from
Epidemic disease measuring method, Western blot, western blot method etc., from can from the aspect of easy and sensitivity detects antibody well,
It is preferred that ELISA method.As ELISA method, competition law, sandwich method etc. can be enumerated.
Here, an example of separation method when showing using Sandwich ELISA.First, identification is present in vesica
After the ligand of the surface antigen on surface is bonded with solid phase carrier, connect the organism sample containing the vesica with bilayer lipid membrane
It touches and forms complex, followed by clean.It is added to monoclonal antibody or its antibody fragment, disease specific memebrane protein
The labelled antibody that matter antibody or its antibody fragment are modified, forms further complex.Then, it is formed by detecting
Complex in labelled amount, the semaphore for coming from the vesica contained by organism sample can be measured, contained by organism sample
Disease specific vesica amount.
(determination method of disease)
The method whether fallen ill of examinee for judging the present invention is characterized in that, comprising using the life from examinee
Object sample, the work being measured using above-mentioned signal measuring method to the signal strength for carrying out the vesica of the above-mentioned complex of self-forming
Sequence (hereinafter also referred to as process (I)).
Then, the signal strength measured in process (I) and the signal strength of the organism sample from collator are carried out
Comparison, it is believed that when the signal strength of the signal intensity ratio collator of examinee is strong, you can be determined as examinee morbidity (hereinafter,
Referred to as process (II)).
The disease of determination method judgement as the disease that can use the present invention, can illustrate Cancerous disease (colorectal cancer, mammary gland
Cancer, carcinoma of endometrium, cervical carcinoma, oophoroma, cancer of pancreas, gastric cancer, cancer of the esophagus, liver cancer, lung cancer, kidney, cutaneum carcinoma etc.), inflammation
The neurologicals such as class disease (rheumatism, osteoarthritis, kidney trouble, pancreatitis, hepatitis, allergy etc.), Alzheimer's disease
Mental diseases, the Parkinson's diseases such as property disease, brain diseases, the relevant disease of immune deficiency, infertility, depression, self-closing disease
Wait refractory diseases, autoimmune disease, circulation system disease, blood disease, disease of digestive system, with aging disease,
Infectious disease etc..Wherein, it is useful to the judgement of Cancerous disease, disease of immune system, particularly especially have to the judgement of Cancerous disease
With.
In addition, if the immunocompetent index applied to cell disorders T cell (CTL), can also apply to cancer
Vaccine judges the effect of Cancerous disease.
Process (I) carries out according to conventional methods in addition to being measured with above-mentioned signal measuring method.
In addition, signal strength of the process (II) for measure in process (I), based on the organism sample from collator
Signal carries out statistics parsing to be compared.Analytic method is not particularly limited, and can use well known method.In addition, its
Judgement afterwards, for example, the organism sample from examinee signal fusing according to the signal of person it is strong when, be judged as morbidity possibility
Property it is high.It should be explained that in the present invention, collator be do not fall ill with the age same sex, others is averaged with examinee, compare
The signal strength of person can be measured together with the signal strength of examinee, and can also use is worth by other measured in advance
The statistical value arrived.
(method of evaluating drug effect of disease curative)
The method of evaluating drug effect of disease curative of the present invention is characterized in that, comprising before being given using disease curative and
The organism sample from examinee after giving, using above-mentioned signal measuring method to carrying out the letter of the vesica of self-forming complex
The process that number intensity is measured (hereinafter also referred to as process (A)).
Then, it is believed that from disease curative give after examinee organism sample in the signal from complex
It, can when intensity is weaker than the signal strength from complex in the organism sample of the examinee before being given from disease curative
To judge that disease curative shows that the possibility of drug effect is high (hereinafter also referred to as process (B)).
As above-mentioned disease curative, the drug of disease that treatment can be judged with above-mentioned determination method can be enumerated.Suitably
Concrete example is anticancer agent, anti-immunity systemic disease medicine.
Process (A) can carry out according to conventional methods other than being measured with above-mentioned signal measuring method.
In addition, process (B) is the signal strength to being measured in process (A), the organism before being given based on disease curative
Signal in sample carries out statistics parsing and is compared.Analytic method is not particularly limited, and can use well known method.
In addition, judgement thereafter, for example, disease curative give after organism sample semaphore than the organism sample before giving
Semaphore it is few when, be judged as that the medical instrument has the possibility for the effect for inhibiting disease high.
Particularly cell, various cancer cells secrete of the excretion body by various cells such as immune system, therefore, it is considered that passing through
The variation for giving excretion body in front and rear blood measured in disease curative (in addition to the increase and decrease of amount, further includes memebrane protein
Amount variation), drug effect in patients can be evaluated.In addition, for example, if combination is for cancer cell specific membrane proteins
Ligand and surface antigen for excretion body ligand, can expect the raising of the specificity of cancer diagnosis, cancer species
It determines, and then the diagnosis medicine to Cancerous disease specificity can be developed.
(kit)
The kit of the present invention is characterized in that having solid phase carrier and fluid composition, and the solid phase carrier is bonded with
Identification is present in the ligand of the surface antigen on the surface of the vesica with bilayer lipid membrane, and the fluid composition contains nonionic
Property surfactant.This kit is to above-mentioned separation method, the judging of above-mentioned disease, the evaluating drug effect of above-mentioned disease curative
It is useful.
As the nonionic surfactant contained by solid phase carrier, fluid composition, can enumerate and the above-mentioned side of separation
The identical substance of the substance that is used in method.The concentration of nonionic surfactant is relative to fluid composition total amount, preferably
0.005~10% (w/v), more preferable 0.02~2% (w/v).In addition, the pH of fluid composition is not particularly limited, preferably
The range of the range of pH5~10, more preferably pH6~8.In order to maintain target pH, usually using buffer solution, for example, can enumerate
Phosphate buffer, three (hydroxymethyl) aminomethane buffer solutions, HEPES buffer solution, MES buffer solutions etc..
(fluid composition)
The fluid composition of the present invention is characterized in that, containing nonionic surfactant, with bilayer lipid membrane
Vesica separation method in use, for cleaning complex, should be included with the separation method of vesica of bilayer lipid membrane:Make
Organism sample containing the vesica with bilayer lipid membrane identifies the surface antigen on the surface for being present in vesica with being bonded with
The solid phase carrier contact of ligand forms the complex formation process of the complex of vesica and solid phase carrier;With cleaning complex
Cleaning process.
The fluid composition phase that the kit of the composition of fluid composition of the present invention, pH and the invention described above has
Together.
The fluid composition of the present invention uses in the separation method of the present invention.
Embodiment
Hereinafter, enumerating embodiment, the present invention is described in detail, and the present invention is not limited to these Examples.
(test example 1 agglomerates validation test (1))
First, prepare buffer solution of the Tris buffered salines (TBS, pH7.4) as control, prepare with a concentration of
The mode of 0.1% (w/v) is by nonionic surfactant (polyoxyethylene (160) polyoxypropylene (30) glycol Pluronic
F-68 (ADEKA, same as below)) it is added to buffer solution of the mixed liquor as embodiment 1 obtained by TBS (pH7.4).
Then, 3 antibody linked magnetic particle of anti-CD 6 is added in a manner of a concentration of 0.1% (w/v) to above-mentioned each buffer solution
(Exosome-Dynabeads Human CD63Isolation/Detection, Life technologies corporations Ref
10606D), which is preserved into the culture supernatant of 100 μ L of buffer solution and HT29 cells containing excretion body (with TBS by 100
Times concentrate dilutes 5 times) 100 μ L add in 1.5mL pipes and are mixed.By it after 25 DEG C are vibrated 1 hour, observe by visual observation
Confirm the condensate of the antibody linked magnetic particle for the internal face for being attached to pipe.Show the result in table 1.
[table 1]
Buffer solution forms | Particle coacervation body | |
Control | TBS | + |
Embodiment 1 | 0.1% (w/v) Pluronic F68, TBS | - |
+:Confirmation particle coacervation body ,-:Particle coacervation body unconfirmed
As shown in table 1, only in TBS during suspended magnetic particle (control), confirm that magnetic particle condensate is attached to inner wall
Face, but add in TBS the nonionic surfactant of 0.1% (w/v) and during suspended magnetic particle (embodiment 1), not really
Recognize the attachment of the condensate.
(test example 2 agglomerates validation test (2))
First, prepare buffer solutions of the TBS (pH7.4) as control, as the buffer solution of embodiment 2~7, prepare to become
The mode of concentration shown in table 2 below is by nonionic surfactant (polyoxyethylene (160) polyoxypropylene (30) glycol
Pluronic F-68) it is added to mixed liquor obtained by TBS (pH7.4), as the buffer solution of comparative example 1 and 2, under becoming
State the concentration shown in table 2 mode bovine serum albumin(BSA) (BSA) is added in TBS (pH7.4) obtained by mixed liquor.
Then, it is added in a manner of a concentration of 0.2% (w/v) to above-mentioned each buffer solution and has been bonded anti-EpCAM antibody (JSR
It is Life Sciences corporations, same as below) magnetic particle (JSR Life Sciences corporations MS300/
It is Carboxyl, same as below), which is preserved to the culture supernatant of 100 μ L of buffer solution and HT29 cells containing excretion body
(100 times of concentrates are diluted 5 times with TBS) 100 μ L are added in 1.5mL pipes and mix.By it after 25 DEG C are vibrated 1 hour,
Observation confirms the condensate of the antibody linked magnetic particle for the internal face for being attached to pipe by visual observation.Show the result in table 2.
[table 2]
Buffer solution forms | Particle coacervation body | |
Control | TBS | + |
Comparative example 1 | 0.1% (w/v) BSA, TBS | + |
Comparative example 2 | 0.1% (w/v) BSA, TBS | + |
Embodiment 2 | 0.02% (w/v) Pluronic F68, TBS | - |
Embodiment 3 | 0.05% (w/v) Pluronic F68, TBS | - |
Embodiment 4 | 0.1% (w/v) Pluronic F68, TBS | - |
Embodiment 5 | 0.2% (w/v) Pluronic F68, TBS | - |
Embodiment 6 | 0.5% (w/v) Pluronic F68, TBS | - |
Embodiment 7 | 1.0% (w/v) Pluronic F68, TBS | - |
+:Confirmation particle coacervation body ,-:Particle coacervation body unconfirmed
As shown in table 2, in anti-EpCAM antibody linked magnetic particle, when suspending only in TBS (control), it is thus identified that magnetic
The condensate of particle is attached to the situation of wall surface.In addition, even if the addition BSA cohesions also do not reduce (Comparative Examples 1 and 2).
On the other hand, (implement when the nonionic surfactant of 0.02~1.0% (w/v) is added in TBS and suspended
Example 2~7), the attachment of the condensate is confirmed.
It should be explained that anti-EpCAM antibody is become into anti-CD9 antibody (Abcam corporations ab2215, same as below) or anti-
CD63 antibody (JSR Life Sciences corporations, same as below), in addition to this, examination is carried out similarly with above-mentioned test example 2
It tests, as a result obtains the result identical with test example 2.
(test example 3 adds influence of the nonionic surfactant to reaction)
It is anti-containing anti-CD9 antibody, anti-CD 63 has been bonded that (i) is separately added into 96 hole white boards (Corning corporations)
The magnetic particle (JSR Life Sciences corporation MS300/Carboxyl) of body, anti-CD81 antibody or anti-EpCAM antibody
TBS, (ii) of 0.05% (w/v) and nonionic surfactant (Pluronic F-68) 1.0% (w/v) use alkaline phosphatase
The culture of MES solution (the 1 μ g/mL), (iii) HT29 cells of the antibody that the enzyme pair antibody identical with above-mentioned (i) is marked
Supernatant (100 times of concentrates are diluted 100 times with TBS) each 25 μ L are mixed.By it in 25 DEG C of vibrations after twenty minutes, side collection
Magnetic side carries out the antibody linked magnetic particles after cleaning reaction using cleaning buffer solution (TBS for containing 0.01% (w/v) Tween20)
Son.50 μ L luminous substrates (LUMIPULSE substrate solutions) are added, after five minutes using luminescence assays machine (Promega corporations
GloMax luminous intensity) is measured.
In addition, to the nonionic surfactant (Pluronic F-68) in the particle suspension by above-mentioned (i)
Concentration changes into 0% (w/v), 0.01% (w/v), 0.02% (w/v), 0.05% (w/v), 0.1% (w/ from 1.0% (w/v)
V), particle obtained from 0.2% (w/v), 0.5% (w/v) preserves buffer solution and also measures luminous intensity as described above.
These results are shown in Fig. 1.
Even if as shown in Figure 1, to particle preserve buffer solution add 0.01~1.0% (w/v) (final concentration 0.0033~
0.33% (w/v)) nonionic surfactant (Pluronic F-68), it is bonded to the 4 kinds of antibody and excretion of magnetic particle
The reaction of body is not also hindered.
(4 cleaning ability validation test of test example)
It is true by the Silver stain of SDS-PAGE when so that the specimen (serum, blood plasma, urine) is reacted with antibody linked magnetic particle
It is cleaned if recognizing and only carrying out particle with TBS (pH7.4), the non-specific adsorption from a specimen can not be substantially reduced.Particularly,
During using serum, non-specific adsorption is more, therefore is confirmed in the following order containing nonionic surfactant using serum
Particle cleaning buffer solution cleaning ability.
First, the magnetism for being bonded anti-CD9 antibody, 3 antibody of anti-CD 6, anti-CD81 antibody or anti-EpCAM antibody will be contained
Particle (JSR Life Sciences corporation MS300/Carboxyl) 0.1% (w/v) and nonionic surfactant
The 150 μ L of TBS (pH7.4) of (Pluronic F-68) 0.1% (w/v) and the 150 μ L of serum from Healthy People are put into 1.5mL pipes
In mixed.
By it after 25 DEG C are vibrated 1 hour, collect magnetic and remove serum deprivation, with cleaning buffer solution ((i) TBS, (ii) of 0.5mL
The TBS of TBS or (iii) containing 0.01% (w/v) Pluronic F-68 containing 0.01% (w/v) Triton X-100
Carry out 2 cleanings.
Then collect magnetic and after cleaning solution is discarded, with 1 × sample buffer (NuPAGE LDS Sample Buffer (4
×), invitrogen Cat no.NP0008 are same as below) 20 μ L suspend antibody linked magnetic particle, stand 5 at 95 DEG C
Minute.By whole amount for SDS-PAGE, Silver stain (Cosmo Bio corporations) is carried out.Show the result in Fig. 2.
As shown in Fig. 2, in all 4 kinds of antibody linked magnetic particles, in cleaning (Fig. 2 merely with TBS:Swimming lane TBS)
In, largely the ingredient non-specific adsorption from serum is in particle, but passes through 0.01% (w/v) of addition in cleaning buffer solution
Nonionic surfactant (Pluronic F-68 or Triton X-100) (Fig. 2:Swimming lane PL, swimming lane TX), it is non-specific
Property absorption drastically reduce.
It should be explained that the nonionic surfactant (Pluronic F-68) of aromatic group is not contained in the molecule
Influence to bilayer lipid membrane is lacked, therefore, it is considered that such nonionic surfactant for not containing aromatic group in the molecule
Agent is particularly suitable for.
(influence of cleaning buffer solution of the test example 5 containing nonionic surfactant to specificity bonding)
In order to which using the cleaning buffer solution containing nonionic surfactant (Pluronic F-68), confirmation is not only
The absorption of non-specific ingredient from a specimen, and even the antigen that specific reaction is carried out with antibody linked magnetic particle is (outer
Secrete body) also do not removed from antibody, the protein detection based on western blot is carried out in the following order.
First, the magnetism for being bonded anti-CD9 antibody, 3 antibody of anti-CD 6, anti-CD81 antibody or anti-EpCAM antibody will be contained
Particle (JSR Life Sciences corporation MS300/Carboxyl) 0.1% (w/v) and nonionic surfactant
The 100 μ L of TBS (pH7.4) of (Pluronic F-68) 0.1% (w/v) and the culture supernatant of the HT29 cells containing excretion body
(100 times of concentrates are diluted 10 times with TBS) 100 μ L are added in 1.5mL pipes and are mixed.
By it after 25 DEG C are vibrated 1 hour, collect magnetic and remove culture supernatant, ((i) contains with the cleaning buffer solution of 0.5mL
Have 0.1% (w/v) BSA PBS or (ii) containing 0.01% (w/v) nonionic surfactant (Pluronic F-
68) TBS) carry out 3 cleanings.
Then collect magnetic after cleaning solution is discarded, antibody linked magnetic particle is suspended with 1 × sample buffer, 20 μ L,
95 DEG C stand 5 minutes.Using after being cleaned using each cleaning buffer solution to each antibody linked magnetic particle of total amount (20 μ L)
Sample, carry out SDS-PAGE.Gel is transferred to pvdf membrane (Trans-Blot Turbo Transfer Pack Midi
Format, 0.2 μm of PVDF (BIO-RAD, Control 400072019)) after, (contain 1% (w/v) BSA with Block buffer
With the TBS of 0.1% (w/v) Tween20) it is vibrated 2 hours at 37 DEG C.It (is contained into 0.1% (w/v) with cleaning buffer solution
The TBS of Tween20) cleaning after, make anti-CD81 antibody (the Clone M38, Abnova as an antibody containing 1 μ g/mL
MAB6435 solution (solvent):TBS containing 0.5% (w/v) BSA) and the HRP as labelled antibody containing 1 μ g/mL
Mark anti-mouse IgG antibody (Mouse TrueBlot ULTRA:Anti-Mouse IgG HRP, Rockland 18-
Solution (solvent 8817-33):TBS containing 0.5% (w/v) BSA) it is reacted 1 hour at 25 DEG C respectively, it is slow with cleaning
After fliud flushing (TBS for containing 0.1% (w/v) Tween20) cleaning, make luminous substrate (SuperSignal West Femto
Maximum Sensitivity substrate, Thermo scientific Cat#34095) reaction, with luminescence assays device
(LAS-3000, FUJIFILM) confirms western blot image.Show the result in Fig. 3.
As shown in figure 3, even if by antibody linked magnetic particle (anti-CD9 antibody, 3 antibody of anti-CD 6, the anti-CD81 after reaction
Antibody, anti-EpCAM antibody) it is cleaned with the TBS containing 0.01% (w/v) nonionic surfactant (Pluronic F-68)
(Fig. 3, swimming lane PLU) compared with the cleaning carried out using the PBS containing 0.1%BSA (Fig. 3, swimming lane BSA), also confirms excretion body
It is not removed from antibody.
(6 stability test of test example)
Even if in order to contain nonionic surfactant when confirming the reaction of antibody linked magnetic particle and when cleaning
(Pluronic F-68) excretion body is also stablized, and measures the size distribution of excretion body in the following order.
First, contain 0.1% (w/v) nonionic surfactant (Pluronic with (i) TBS (pH7.4) or (ii)
The culture supernatant (100 times of concentrates) of HT29 cells is diluted 10 times of (10 × culture supernatants by TBS (pH7.4) F-68)
Liquid), stored refrigerated 3 days.Thereafter, 10 × culture supernatant TBS is diluted 100 times, uses NanoSight LM10 (NANO
SIGHT companies) confirm excretion body size distribution.
Even if by excretion body the nonionic surfactant (Pluronic F-68) containing 0.1% (w/v) TBS
Middle preservation compared with only with the preservation of TBS, also can't see the variation of the size distribution of excretion body.Even if it is therefore contemplated that in antibody
Addition nonionic surfactant (Pluronic F-68) in the preservation buffer solution and cleaning buffer solution of magnetic particle is bonded,
The stability of excretion body is not also influenced.
(7 nonionic surfactant of test example)
Influence of the various nonionic surfactants in reaction shown in for confirmation form 3, is surveyed in the following order
The reactivity of antibody linked magnetic particle and excretion body in the presence of fixed each nonionic surfactant.
First, (i) is contained into magnetic particle (the JSR Life Sciences corporations for being bonded anti-CD81 antibody
MS300/Carboxyl) the TBS (pH7.4) of 0.05% (w/v) and the nonionic surfactant 1.0% (w/v) shown in table 3
200 μ L or (ii) contain magnetic particle (the JSR Life Sciences corporations MS300/ for being bonded anti-CD81 antibody
Carboxyl) the PBS200 μ L of 0.05% (w/v) and BSA0.1% (w/v), in the culture with the HT29 cells containing excretion body
Clear liquid (100 times of concentrates are diluted 10 times with TBS) 10 μ L are added in 1.5mL pipes and are mixed, vibrated 1 hour at 25 DEG C.
It is added into 50 μ L into 96 hole white boards (Corning corporations), cleaning buffer solution is used (to contain in Ji Ci
The TBS of 0.01% (w/v) Tween20) the antibody linked magnetic particle after reaction is cleaned.It is added in thereto with alkaline phosphorus
MES solution (1 μ g/mL) 50 μ L that the antibody of anti-CD81 antibody is marked in sour enzyme are mixed.
By it in 25 DEG C of vibrations after twenty minutes, cleaning buffer solution is used (to contain 0.01% (w/v) Tween20 in Ji Ci
TBS) the antibody linked magnetic particle after reaction is cleaned.Add 50 μ L luminous substrates (FUJIREBIO systems,
LUMIPULSE substrate solutions), after five minutes luminous intensity is measured using luminescence assays machine (Promega corporation GloMax).It will
As a result it is shown in table 4.
As shown in table 4, even if addition polyalkylene glycol ethylene oxide adduct (Pluronic F-68, Pluronic
L-62) and the aliphatic ester of sorbitan (Tween20, Tween80), also can't see caused by the destruction of excretion body with it is anti-
The reduction of the reactivity of body bonding magnetic particle.On the other hand, for living as the non-ionic surface containing aromatic group
Property the TritonX-100 (HLB value 13.5) of agent and Nonidet P-40 (HLB value 13.1), it is thus identified that think the broken of excretion body
The reduction of the bad reactivity for reason.
Even if according to the above, add nonionic in thinking the preservation buffer solution and cleaning buffer solution of antibody linked magnetic particle
Property surfactant (not containing aromatic group in the molecule), stability to excretion body and with antibody linked magnetic particle
Reactivity does not also influence.
[table 3]
[table 4]
(test example 8 captures the confirmation of vesica)
In order to confirm that the excretion body captured with antibody linked magnetic particle is not destroyed, particle size distribution meter and transmission-type are used
Electron microscope measures the grain size and form of excretion body in the following order.
To contain whole particle concentration be 0.1% (w/v) be bonded anti-CD9 antibody, 3 antibody of anti-CD 6, anti-CD81 antibody or
The magnetic particle (JSR Life Sciences corporation MS300/Carboxyl) of person's anti-EpCAM antibody and 0.1% (w/v's)
The 100 μ L of TBS (pH7.4) of nonionic surfactant (Pluronic F-68), with the HT29 cells containing excretion body
100 μ L of culture supernatant are added in 1.5mL pipes and are mixed.By it after 25 DEG C are vibrated 1 hour, collect magnetic and remove culture supernatant
Liquid carries out 3 cleanings with the cleaning buffer solution (TBS for containing 0.1% (w/v) Pluronic F-68) of 0.5mL.
Collect magnetic and remove cleaning buffer solution, with excretion body elution buffer (JSR Life Sciences corporations
ExoCapTMExosome Isolation and Enrichment Kits Exosome Elution Buffer) by antibody key
It closes magnetic particle to suspend, prepares elution excretion body.Thereafter, with nano-particle resolver (NanoSight LM10, NANO
SIGHT companies) measure the size distribution for eluting excretion body.Show the result in Fig. 4 and Fig. 5.It should be explained that Fig. 4 is using anti-
The particle size distribution figure of elution excretion body during CD63 antibody linked magnetic particles.
As a result, it confirmed the waveform (Fig. 4) in 100nm or so size distributions with peak.It is in addition, anti-with being bonded
CD9 antibody, 3 antibody of anti-CD 6, anti-CD81 antibody or anti-EpCAM antibody whole elution excretion bodies for capturing of particle in confirm
Identical size distribution (Fig. 5).
Using the magnetic particle for being bonded anti-CD9 antibody, directly observed as described above using transmission electron microscope
The form of elution excretion body prepared by ground.Negative staining is carried out using phosphotungstic acid or acetic acid uranium.Show the result in Fig. 6.
As a result, it confirmed the bilayer lipid membrane (arrow in Fig. 6 of structure spherical as vesica (Fig. 6) and vesica
Head).
According to result above, the vesica captured with each antibody linked magnetic particle is similary with the grain size of excretion body reported
Ground has the grain size of 100nm or so, and form is also consistent with the observation result reported, therefore, it is considered that being excretion body, by catching
It catches process and cleaning process excretion body is not destroyed.
(detection of the nucleic acid in 9 vesica of test example)
The nucleic acid (full RNA) in the vesica that is captured with antibody linked magnetic particle is carried out using microchip type electrophoretic apparatus
Detection.As the standard excretion body that sample uses, excretion body is detached from the culture supernatant of HT29 cells using ultracentrifugation, is used
After PBS adjustment, protein quantification (BIO-RAD DC Protein Assay) is carried out.Specific sequence described below.
To contain whole particle concentration be 0.1% (w/v) be bonded anti-CD9 antibody, 3 antibody of anti-CD 6, anti-CD81 antibody or
The magnetic particle (JSR Life Sciences corporation MS300/Carboxyl) of person's anti-EpCAM antibody and 0.1% (w/v's)
The 100 μ L of TBS (pH7.4) of nonionic surfactant (Pluronic F-68), with containing standard excretion body (0 μ g, 1 μ g,
3 μ g or 10 μ g) PBS100 μ L add in 1.5mL pipes in mixed.By it after 4 DEG C are vibrated about 18 hours, collect magnetic and remove
Unreacted excretion body is removed, is carried out 3 times with the cleaning buffer solution (TBS for containing 0.1% (w/v) Pluronic F-68) of 1mL
Cleaning collects magnetic and removes cleaning buffer solution.
Using RIP-Assay Kit for microRNA (MBL corporations, with reference to two-step method), from antibody linked magnetic
Property the excretion body that captures of particle in after extraction whole RNA, with RNase-free water adjustment.
Using microchip type electrophoretic apparatus Biochemical Analyzer (Agilent Technologies systems) to come antibody key of using by oneself
The whole RNA for closing the excretion body that magnetic particle captures are detected.Show the result in Fig. 7.
As shown in fig. 7, confirm the peak of Small RNA and mRNA etc..In addition, the amount of the nucleic acid of detection and the standard excretion scale of construction
Proportionally increase.
(detection of the microRNA in 10 vesica of test example)
Using quantitative PCR method, in the following order to the nucleic acid in the vesica that is captured with antibody linked magnetic particle
(microRNA) it is measured.
First, as antibody linked magnetic particle, (i) anti-CD 63 for the use of whole particle concentration being respectively 0.1% (w/v) resists
Body is bonded magnetic particle or (ii) anti-CD9 antibody linkeds magnetic particle, 3 antibody linked magnetic particle of anti-CD 6, anti-CD81 antibody
It is bonded the mixture (mass ratio 1 of magnetic particle and anti-EpCAM antibodies Antibodies bonding magnetic particle:1:1:1) it, in addition to this, uses
The method extraction whole RNA identical with test example 9.Then, using miScript II RT kit (QIAGEN corporations) and
MiScript SYBR Green PCR kit (QIAGEN corporations) quantify microRNA (let-7a-1).It will knot
Fruit is shown in Fig. 8.
As a result, amplification curve as shown in Figure 8 is obtained, it can be from the excretion physical examination captured with antibody linked magnetic particle
Measure the microRNA of specificity.The various microRNA from vesica are determined using antibody linked magnetic particle as a result,
Amount can be used in the judgement of disease by analysis.
(detection of protein of the test example 11 from vesica)
Using western blot method to existing for the inside of the vesica captured with antibody linked magnetic particle and surface come
It is detected from the protein of vesica.
First, the magnetism for being bonded anti-CD9 antibody, 3 antibody of anti-CD 6, anti-CD81 antibody or anti-EpCAM antibody will be contained
Particle (JSR Life Sciences corporation MS300/Carboxyl) 0.1% (w/v) and nonionic surfactant
The 100 μ L of TBS (pH7.4) of (Pluronic F-68) 0.1% (w/v), the culture supernatant with the HT29 cells containing excretion body
Liquid (100 times of concentrates are diluted 10 times with TBS) 0.1mL is added in 1.5mL pipes and is mixed.
By it after 25 DEG C are vibrated 1 hour, collect magnetic and remove culture supernatant, (contained with the cleaning buffer solution of 0.5mL
The TBS of 0.1% (w/v) nonionic surfactant (Pluronic F-68)) carry out 3 cleanings.
Then collect magnetic after cleaning solution is discarded, antibody linked magnetic particle is suspended with 1 × sample buffer, 20 μ L,
95 DEG C stand 5 minutes.It should be explained that the detection of surface protein (CD9, CD63, CD81, EpCAM) is carried out with non-reduced processing,
The detection of inside protein (Alix, Hsp70) is carried out with reduction treatment.Using each sample of whole amount (20 μ L), SDS- is carried out
PAGE.After gel is transferred on pvdf membrane, (contain 1% (w/v) BSA's and 0.1% (w/v) Tween20 with Block buffer
TBS it) is vibrated 2 hours at 37 DEG C.It with cleaning buffer solution (TBS for containing 0.1% (w/v) Tween20) is cleaned, makes one respectively
Secondary antibody and labelled antibody react 1 hour at 25 DEG C.
It should be explained that the detection of the protein other than Hsp70, using anti-CD9 antibody (Abcam corporation ab2215), resists
CD63 antibody (MX-49.129.5, Mouse IgG1, SantaCruz sc-5275), anti-CD81 antibody (Clone M38,
Abnova MAB6435), anti-EpCAM antibody (JSR Life Sciences corporations), anti-Alix antibody (Alix (3A9)
Mouse mAb, Cell signaling technology#2171S) as an antibody, resisted using HRP labels anti-mouse IgG
Body (Mouse TrueBlot ULTRA:Anti-Mouse IgG HRP, Rockland 18-8817-33) it is anti-as label
Body.In addition, the detection of Hsp70 uses Anti-HSP70, Rabbit-Poly, with HRP Conjugated Secondary
Antibody (SBI corporations, EXOAB-Hsp70A-1).
An above-mentioned antibody and labelled antibody (contain 0.1% (w/v) Tween20 after reaction, with cleaning buffer solution
TBS) cleaning, make luminous substrate (SuperSignal West Femto Maximum Sensitivity substrate,
Thermo scientific Cat#34095) reaction, confirm Western with luminescence assays device (LAS-3000, FUJIFILM)
Print image.As a result it is shown in Fig. 9.
As a result, in whole antibody linked magnetic particles, detected and come from target size from the excretion body of capture
The surface protein (CD9, CD63, CD81, EpCAM) of vesica and the band (Fig. 9) of inside protein (Alix, Hsp70).
In addition, by the culture supernatant 0.1mL for the HT29 cells that above-mentioned reaction uses become respectively standard excretion body (1,3,
10 μ g), the culture supernatant 1mL of HT29 cells, utilize the amount (table of protein of the method detection from vesica same as described above
Face protein C D9).Anti- CD9 antibody will be used to be shown in Figure 10 as a result during antibody.
As a result, the amount of the protein from vesica of detection and the standard excretion scale of construction (1,3,10 μ g) reacted and culture
The liquid measure (0.1,1mL) of supernatant proportionally increases (Figure 10).Therefore, by being quantified to the protein from vesica,
The amount of the vesica in body fluid or culture supernatant can be measured.
(detection of vesica of the test example 12 from body fluid and culture supernatant)
Using western blot method to coming from the vesica in various cell culture supernatants and body fluid (serum, blood plasma, urine)
Protein be detected.It should be explained that using 3 specimen of Healthy Human Serum (Serum A, Serum B, Serum C) as blood
Clearly, using 3 specimen of Healthy People heparin blood plasma (Plasma (Heparin) A, Plasma (Heparin) B, Plasma (Heparin)
C) as blood plasma, using 3 specimen of healthy human urine (Urine D, Urine E, Urine F) as urine.It is described below specific suitable
Sequence.
First, by (i) containing being bonded anti-CD9 antibody, 3 antibody of anti-CD 6, anti-CD81 antibody or anti-EpCAM antibody
Magnetic particle (JSR Life Sciences corporation MS300/Carboxyl) 0.1% (w/v) and nonionic surfactant
The 100 μ L of TBS (pH7.4) of (Pluronic F-68) 0.1% (w/v), with (ii) cell culture supernatant (HT-29,293T
Or NCI-H520) or from Healthy People body fluid (serum, blood plasma or urine (each 3 specimen)) 100 μ L add in 1.5mL pipes in into
Row mixing.
By it in 25 DEG C of vibrations after twenty minutes, collect magnetic and remove unreacted liquid, (contained with the cleaning buffer solution of 0.5mL
The TBS of 0.1% (w/v) nonionic surfactant (Pluronic F-68)) carry out 3 cleanings.
Then collect magnetic after cleaning solution is discarded, antibody linked magnetic particle is suspended with 1 × sample buffer, 20 μ L,
95 DEG C stand 5 minutes.Using each sample of whole amount (20 μ L), SDS-PAGE is carried out.After gel is transferred on pvdf membrane,
It is vibrated 2 hours at 37 DEG C with Block buffer (TBS for containing 1% (w/v) BSA and 0.1% (w/v) Tween20).By it with clearly
Wash buffer (TBS for containing 0.1% (w/v) Tween20) is cleaned.
The anti-CD9 antibody as antibody and the HRP as labelled antibody is made to mark anti-mouse IgG antibody respectively
(Mouse TrueBlot ULTRA:Anti-Mouse IgG HRP, Rockland 18-8817-33) it is small in 25 DEG C of reactions 1
When, after it is cleaned with cleaning buffer solution (TBS for containing 0.1% (w/v) Tween20), react luminous substrate, with hair
Photo-detector (LAS-3000, FUJIFILM) confirms western blot image.Show the result in Figure 11.
As a result, in the magnetic particle for being bonded anti-CD9 antibody, 3 antibody of anti-CD 6, anti-CD81 antibody, detect and come from
The surface protein (CD9) (Figure 11) of vesica captured from whole culture supernatant and body fluid.On the other hand, it has been bonded anti-
In the magnetic particle of EpCAM antibody, the culture supernatant of celliferous HT29 and NCI-H520 is produced from EpCAM, detects and comes from
The surface protein (CD9) of vesica, but from the EpCAM celliferous 293T of non-production and whole Healthy People body fluid (serum, blood plasma,
Urine) surface protein (CD9) (Figure 11) from vesica is not detected.
Therefore, by using the particle for being bonded the ligand for antigens from disease such as cancer markers, Neng Goufen
It is detected from from Healthy People and from the vesica for suffering from patient.As a result, by will the signal from the organism sample of examinee
Intensity and the signal strength of the organism sample from Healthy People compare, and can determine that examinee falls ill.
(detection of vesica of the test example 13 based on ELISA)
Quantitative detection is carried out to the signal strength from vesica using ELISA method.Specific sequence described below.
25 μ L are separately added into 96 hole white boards (Corning corporations) and contain that be bonded with anti-CD9 antibody, anti-CD 63 anti-
The magnetic particle (JSR Life Sciences corporation MS300/Carboxyl) of body, anti-CD81 antibody or anti-EpCAM antibody
The TBS (pH7.4) and cell of 0.1% (w/v) and nonionic surfactant (Pluronic F-68) 0.1% (w/v)
The culture supernatant of (293T, NCI-H520, HT29 or 22Rv-1) is simultaneously mixed.It is vibrated 20 minutes at 25 DEG C
Afterwards, use cleaning buffer solution (TBS for containing 0.01% (w/v) Tween20) magnetic to the antibody linked after reaction in Ji Ci
Particle is cleaned.Cleaning buffer solution is removed in Ji Ci, the anti-CD9 antibody of addition alkali phosphatase enzyme mark or anti-CD81 resist
MES solution (0.5 μ g/mL) 50 μ L of the antibody of body.By it in 25 DEG C of vibrations after twenty minutes, cleaning buffer solution is used in Ji Ci
(TBS for containing 0.01% (w/v) Tween20) cleans the antibody linked magnetic particle after reaction.50 μ L are added to shine
Substrate (FUJIREBIO systems, LUMIPULSE substrate solutions), after five minutes using luminescence assays machine (Promega corporations
GloMax luminous intensity) is measured.
It thereafter, will be to the signal strength for the vesica measure for coming from cancer cell (NCI-H520, HT29,22Rv-1) and to coming
The signal strength measured from the vesica of 293T cells is compared.Show the result in table 5 and 6.
As a result, being bonded in the magnetic particle of anti-CD9 antibody, 3 antibody of anti-CD 6 or anti-CD81 antibody, marked based on ALP
Remember the detection (table 5) of anti-CD9 antibody and the detection of anti-CD81 antibody (table 6) is marked to be from the outer vesica of cancer cell based on ALP
Signal strength approached with the signal strength from the extracellular vesicas of 293T.On the other hand, it has been bonded the magnetism of anti-EpCAM antibody
In particle, the signal strength of vesica is significantly higher than the signal strength from the extracellular vesicas of 293T outside cancer cell.
In addition, for be bonded the magnetic particle of anti-CD9 antibody, 3 antibody of anti-CD 6 or anti-CD81 antibody capture it is outer
The ratio of body, the catch rate height based on anti-CD81 antibody linkeds magnetic particle of the extracellular vesicas of NCI-H520 etc. are secreted, because cancer is thin
The type of born of the same parents and it is different (table 5, table 6).
Therefore, by using being bonded for common labelled protein (CD9, CD63, CD81 etc.) and cancer from vesica
The particle of the ligand of the antigen of disease marker etc. (EpCAM etc.) from disease, can will be from Healthy People and from suffering from patient's
Vesica detaches, and quantitative detection is carried out using ELISA method etc..As a result, by will the signal from the organism sample of examinee it is strong
The signal strength of degree and the organism sample from Healthy People compares, and is determined as that examinee falls ill.
In addition, by the organism sample from examinee before being given by disease curative and after giving, with identical
Method is detected the signal strength from vesica, compares, and can evaluate the drug effect of disease curative.
[table 5]
The comparison (/ vesica from 293T of signal strength based on ALP label CD9 antibody
[table 6]
The comparison (/ vesica from 293T of signal strength based on ALP label CD81 antibody
Claims (13)
1. a kind of separation method of the vesica with bilayer lipid membrane, which is characterized in that including:
Complex formation process makes the organism sample containing the vesica with bilayer lipid membrane and is bonded with the particle shape of ligand
Solid phase carrier contacted, form the complex of the vesica and the solid phase carrier, wherein, ligand identification is present in
The surface antigen on the surface of the vesica;
Cleaning process cleans the complex;
It does not contain in the molecule and the complex is carried out in the presence of the nonionic surfactant of aromatic group forms work
At least complex formation process in sequence and the cleaning process.
2. separation method according to claim 1, wherein, the nonionic surfactant is selected from polyalkylene two
Alcohol ethylene oxide adduct, the aliphatic ester of sorbitan, the aliphatic ester of D-sorbite, sorbitan fatty acid ester
1 kind in the ethylene oxide adduct of ethylene oxide adduct and sorbitan fatty acid ester or two or more.
3. separation method according to claim 1, wherein, the nonionic surfactant is selected from polyalkylene two
1 kind or 2 kinds in the ethylene oxide adduct of alcohol ethylene oxide adduct and sorbitan fatty acid ester.
4. separation method according to claim 1, wherein, the nonionic surfactant is the poly- of block copolymerization type
Aklylene glycol ethylene oxide adduct.
5. separation method according to claim 1, wherein, the nonionic surfactant is with by polycyclic oxygen second
The block copolymerization of block and the block for being more than 3 by the carbon atom number of the alkylidene that polyoxyalkylene is formed that alkane is formed
Object.
6. separation method according to claim 1, wherein, the organism sample is body fluid or cells and supernatant
Liquid.
7. separation method according to claim 1, wherein, the vesica is excretion body.
8. separation method according to claim 7, wherein, the surface antigen is the antigen egg for being present in excretion body surface face
White matter, the ligand are the antibody for the antigen protein that identification is present in excretion body surface face.
9. separation method according to claim 1, wherein, the solid phase carrier is magnetic particle.
10. separation method according to claim 9, wherein, the cleaning process, which includes, utilizes magnetic force by the magnetic particles
Son aggregation and by the collection magnetic process of magnetic particle and liquid phase separation and the magnetic particle detached in the collection magnetic process is made to be dispersed in clearly
Dispersion step in washing lotion.
A kind of 11. detection method of the nucleic acid in vesica, which is characterized in that the separation in claim 1~10 described in wantonly 1
After method, the detection of nucleic acids process being detected to the nucleic acid in vesica is further included.
12. a kind of detection method of the protein from vesica, which is characterized in that in claim 1~10 described in wantonly 1
After separation method, the egg being detected to the protein for being present in the inside of vesica and at least one party on surface is further included
White matter detects process.
A kind of 13. signal measuring method from vesica, which is characterized in that the separation in claim 1~10 described in wantonly 1
After method, the signal measuring work that the signal strength of the vesica to carrying out complex described in self-forming is measured is further included
Sequence.
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US20200080997A1 (en) * | 2016-12-05 | 2020-03-12 | The Penn State Research Foundation | Lipid-based probes for extracellular isolation |
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US20210396633A1 (en) * | 2018-10-17 | 2021-12-23 | H.U. Group Research Institute G.K. | Method for recovering extracellular vesicles |
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